As one skilled in the art will appreciate, there has been a long felt need to substantially reduce the cost of fabricating microelectrode arrays (MEAs) for cell-based biosensors, etc. The present invention provides a method to fabricate low-cost microelectrode arrays and corresponding circuitry. More specifically, the present invention takes advantage of printed circuit board (PCB) fabrication technology to enable the efficient mass production of low-cost microelectrode arrays.
A key of the present invention is the discovery that certain unmodified PCB processes, combined with selective materials, can be utilized to mass-produce microelectrode arrays suitable for biological applications. As an example, sixty-electrode arrays can be simultaneously fabricated on one substrate.
PCB materials and fabrication processes are commonly used to produce computer motherboards and the likes. Conventional PCBs are not suitable for biological applications because many standard materials and processes have poor biocompatibility. In particular, standard, unmodified PCB fabrication technology is not generally known to be useful or viable for fabricating microelectrode arrays for recording extracellular electrical signals from electrically active cell cultures.
A conventional PCB is made of conductive wires (traces) “printed” or otherwise attached to a sheet of insulator (substrate). The PCB substrate is typically made of a phenol formaldehyde resin or a fiberglass-reinforced epoxy composite material.
There are three common PCB fabrication methods: photoengraving, PCB milling, and PCB printing. Although a conventional PCB can be made by adding traces to the substrate, the vast majority of conventional PCBs are manufactured by gluing a layer of copper foil over the entire PCB substrate, sometimes on both sides, then removing unwanted copper, leaving only the copper traces. A conventional PCB can also be made with a trace layer inside, producing a multi-layer PCB. After a conventional PCB is manufactured, components are typically attached to the traces by soldering.
U.S. Pat. No. 6,024,702, issued to Iversen and entitled, “IMPLANTABLE ELECTRODE MANUFACTURED WITH FLEXIBLE PRINTED CIRCUIT,” discloses an implantable cylindrical electrode for monitoring tissue electrical activity and for tissue electrical stimulation. Iversen's implantable electrode is made with a printed circuit etched onto a flexible, non-conducting backing material of mylar or silicone. This patent addresses the problem of recording brain electrical activity at epileptogenic foci, which may comprise thousands or tens of thousands of neurons. It does not address the problem of recording extracellular electrical signals from electrically active cell cultures in vitro.
The extracellular electrical recording of electrogenic cells cultured over microelectrode arrays (MEAs) is a technique used increasingly over the last decade. As a fundamental research tool, it has been shown to yield valuable information on neuronal network and cardiac tissue dynamics. Recent reviews in the relevant field provide numerous references and further illustrate the applications of microelectrode arrays in cell-based biosensors, drug discovery, and safety pharmacology.
Production of these microelectrode arrays has typically relied on thin-film technologies derived from the microelectronic manufacturing industry. These technologies enable high-resolution (electrodes smaller than 10 μm) and high-density arrays (typically 32 to 64 electrodes with spacing down to 100 μm). However, none of these technologies are truly standard, resulting in high processing costs.
Scalability is another issue, as the cost of the chips increases markedly with size, as do packaging costs with array element number. This is of particular importance for multi-well designs incorporating several arrays. Lastly, the current paradigm is to reuse MEAs multiple times, driven mainly by the cost of commercially available MEAs. In addition to concerns about degradation of the array and of its performance and cross-contamination between experiments, such recycling involves additional (and often underestimated) costs due to handling, cleaning, and inspection.
The present invention addresses the aforementioned problems with a fabrication method based on unmodified PCB technology for producing low-cost MEAs useful for many practical applications, especially disposable applications such as cell-based biosensors, drug discovery, etc.
According to the present invention, unmodified, carefully selected PCB fabrication processes are used to simultaneously fabricate a plurality of microelectrode arrays and sensors on a suitable substrate made of, for instance, polymer. Depending upon the design and/or application, the microelectrode arrays and sensors can be formed on one side or both sides of the substrate. The substrate accordingly contains patterned conductive traces on one side or both sides. In an embodiment, the sensor is an integrated temperature sensor/heater.
The integrated heater/sensor takes advantage of the thermal properties of the metal lines “printed” on the substrate. Heater or heating elements can be incorporated on the same substrate using metal (e.g., copper) traces with minimal line width, providing fast, low power, controlled heating of the substrate. Similarly, temperature sensors or sensing elements can be realized using metal traces with minimal line width, providing a direct and accurate measurement and control of the substrate temperature.
In a specific embodiment, both the microelectrode array and the integrated sensor/heater are centrally located within the overall dimensions or footprint of a PCB. Covering layers (coverlays or insulation layers) on both sides insulate the metal line and defines the electrodes. The locations of the MEAs and sensors are not to be construed as limiting. For example, the fabrication method according to the present invention can be used to fabricate a disposable cartridge that has a plurality of MEAs (e.g., an array of microelectrode arrays) and sensors. In this case, other arrangements are possible and the MEAs and sensors do not need to be centrally located within the overall dimensions of the cartridge.
Based on the principles disclosed herein, multiple sensors of various kinds can be integrated onto the substrate. As an example, an oxygen sensor structure comprising three gold-plated electrodes is defined using the standard, unmodified PCB fabrication process. A solid electrolyte and a passivation layer are then deposited and patterned on top of the oxygen sensor electrodes, by way of, for instance, screen-printing, spraying, or droplets dispensing methods. Other possible sensor or sensors might measure pH, glucose, dopamine, or use a variety of ion selective amperometric techniques well known in the art.
An embodiment of the present invention is particularly useful for recording extracellular electrical signals from electrically active cell cultures. Examples of electrically active cells include, but not limited to, cardiomyocytes, neurons, pancreatic cells, and the likes. In the case for cardiac myocyte cultures, the electrode detects a traveling wave resulting from the depolarization of multiple, synchronized cells in a syncytium. Non-electrically active cells can also be utilized for toxin detection, pharmaceutical screening and the like, but measuring impedance of the cells and medium using the MEA. Such methods are well-known in the art, see, for example, U.S. Pat. No. 5,981,268, issued to Kovacs et al. and entitled, “Hybrid Biosensors,” disclosing cell-based biosensors including variants with genetically engineered cells; Borkholder, D. A., Maluf, N. I., and Kovacs, G. T. A., “Impedance Imaging for Hybrid Biosensor Applications,” Proceedings, Solid-State Sensor and Actuator Workshop, Hilton Head, S.C., Jun. 3-6, 1996, pp. 156-160; C. R. Keese and I. Giaever, “A whole cell biosensor based on cell-substrate interactions,” Annual Intl. Conf. IEEE Engineering in Medicine and Biology Society, 12, 2 (1990), pp. 500-501; C. R. Keese and I. Giaever, “A biosensor that monitors cell morphology with electrical fields,” IEEE Engineering in Medicine and Biology, 13, 3 (1994), pp. 402-408; and C. Xiao and J. H. Luong, “On-line monitoring of cell growth and cytotoxicity using electric cell-substrate impedance sensing (ECIS),” Biotechnology Progress, 19, 3 (2003), pp. 1000-1005.
Still further objects and advantages of the present invention will become apparent to one skilled in the art upon reading and understanding the preferred embodiments described below with reference to the following drawings.